The overall goal of this proposal is to define the contribution of posttranscriptional control to herpes simplex virus type 1 (HSV-1) gene regulation and to elucidate the role of ICP27 in posttranscriptional processes. ICP27 is an immediate-early protein which is required for productive infection. It has been shown to have a multitude of effects on gene expression including a contribution to host shutoff, regulation of immediate-early and early gene products, induction of late gene products and a stimulation of viral DNA replication. Recent studies from our laboratory have shown that ICP27 performs these functions at least in part posttranscriptionally. These studies further showed that ICP27 may affect pre-mRNA processing, specifically polyadenylation and splicing. In this proposal we will probe whether ICP27 participates in these processes and if so, how, and we will assess the overall effect on HSV-1 regulation. First, the specific stage in RNA metabolism affected by ICP27 will be determined by detailed comparisons of RNA synthesis, RNA processing, and RNA nuclear and cytoplasmic accumulation in wild-type and ICP27 mutant-infected cells. The data thus far strongly suggest that ICP27 affects RNA processing. To determine if ICP27 influences poly(A) site usage, and to identify potentially responsive signals, a mutational analysis will be performed on two poly(A) sites which were present in target genes activated by ICP27. To evaluate the importance of regulated poly(A) site usage during HSV-1 infection, poly(A) site swapping will be done. Chimeric genes, under early or late promoter control will be constructed with selected poly(A) sites from immediate-early, early and late genes. These will be introduced into the viral genome in wild-type and ICP27 mutant backgrounds. The pattern of accumulation of poly(A+) RNA will be compared to see if altering the poly(A) site will change the time of appearance of transcripts under identical promoter control. To analyze the effect of ICP27 on splicing, reporter genes containing introns will be recombined into the genome in wild-type and ICP27 mutant viruses, and the appearance of correctly spliced RNAs will be monitored throughout infection. Immunofluorescence studies will also be performed with anti-snRNP antisera in wild-type and ICP27 mutant infections to monitor any changes in splicing complexes. To probe the mechanism by which ICP27 acts, nuclear extracts competent for splicing or polyadenylation will be used to directly analyze the effect of ICP27 on the efficiency and specificity of these processes. To identify potential molecular interactions, RNA binding studies will be performed by U.V. cross-linking and by gel shift analysis. Protein-protein interactions will be probed by fractionation studies, coimmunoprecipitation, affinity chromatography, and chemical cross-linking studies. Structure-function correlations will be made using our battery of ICP27 mutants as well as additional mutants to be made in regions likely to be involved in RNA or protein interactions. These studies are important because accumulating data suggest that the expression of many cellular and viral genes is regulated posttranscriptionally. Few factors which affect the efficiency and specificity of RNA processing have been Identified. The possible participation of ICP27 in both splicing and polyadenylation could uncover factors which link these complex processes.